Humanised anti-mag antibody or functional fragment thereof

ABSTRACT

The present invention relates to altered antibodies to myelin associated glycoprotein (MAG), pharmaceutical formulations containing them and to the use of such antibodies in the treatment and/or prophylaxis of neurological diseases/disorders.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage entry ofPCT/EP03/08749, filed 5 Aug. 2003, and claims the benefit of priority ofUnited Kingdom 0218230.1, filed 6 Aug. 2002, United Kingdom 0218232.7,filed 6 Aug. 2002, United Kingdom 0218234.3, filed 6 Aug. 2002, andUnited Kingdom 0218229.3, filed 6 Aug. 2002.

FIELD OF THE INVENTION

The present invention relates to altered antibodies that bind to myelinassociated glycoprotein (MAG) and neutralise the function thereof,polynucleotides encoding such antibodies, pharmaceutical formulationscontaining said antibodies and to the use of such antibodies in thetreatment and/or prophylaxis of neurological diseases. Other aspects,objects and advantages of the present invention will become apparentfrom the description below.

BACKGROUND OF THE INVENTION

Stroke is a major cause of death and disability in the Western World.There is no approved therapy for the treatment of stroke other than t-PAwhich has to be administered within 3 h of onset following a CT scan toexclude haemorrhage. To date most therapeutic agents directed towardsthe treatment of acute stroke (i.e. neuroprotection), have predominantlyinvolved targeting glutamate receptors and their down stream signallingpathways known to be involved in acute cell death. However to date thesestrategies have proved unsuccessful in clinical trials and are oftenassociated with dose-limiting side effects (Hill & Hachinski, TheLancet, 352: (suppl III) 10-14 (1998)). Therefore there is a need fornovel approaches directed towards the amelioration of cell deathfollowing the cessation of blood flow.

Following the onset of stroke, some degree of spontaneous functionalrecovery is observed in many patients, suggesting that the brain has the(albeit limited) ability to repair and/or remodel following injury.Agents that have the potential to enhance this recovery may thereforeallow intervention to be made much later (potentially days) followingthe onset of cerebral ischaemia. Agents which are able to offer bothacute neuroprotection and enhance functional recovery may providesignificant advantages over current potential neuroprotectivestrategies.

The mechanisms underlying functional recovery are currently unknown. Thesprouting of injured or non-injured axons has been proposed as onepossible mechanism. However, although in vivo studies have shown thattreatment of spinal cord injury or stroke with neurotrophic factorsresults in enhanced functional recovery and a degree of axonalsprouting, these do not prove a direct link between the degree of axonalsprouting and extent of functional recovery (Jakeman, et al. 1998, Exp.Neurol. 154: 170-184, Kawamata et al. 1997, Proc Natl Acad. Sci. USA.,94:8179-8184, Ribotta, et al. 2000, J Neurosci. 20: 5144-5152).Furthermore, axonal sprouting requires a viable neuron. In diseases suchas stroke which is associated with extensive cell death, enhancement offunctional recovery offered by a given agent post stroke may thereforebe through mechanisms other than axonal sprouting such asdifferentiation of endogenous stem cells, activation of redundantpathways, changes in receptor distribution or excitability of neurons orglia (Fawcett & Asher, 1999, Brain Res. Bulletin, 49: 377-391, Horner &Gage, 2000, Nature 407 963-970).

The limited ability of the central nervous system (CNS) to repairfollowing injury is thought in part to be due to molecules within theCNS environment that have an inhibitory effect on axonal sprouting(neurite outgrowth). CNS myelin is thought to contain inhibitorymolecules (Schwab M E and Caroni P (1988) J. Neurosci. 8, 2381-2193).Two myelin proteins, myelin-associated glycoprotein (MAG) and Nogo havebeen cloned and identified as putative inhibitors of neurite outgrowth(Sato S. et al (1989) Biochem. Biophys. Res. Comm. 163,1473-1480;McKerracher L et al (1994) Neuron 13, 805-811; Mukhopadhyay G et al(1994) Neuron 13, 757-767; Torigoe K and Lundborg G (1997) Exp.Neurology 150, 254-262; Schafer et al (1996) Neuron 16, 1107-1113;WO9522344; WO9701352; Prinjha R et al (2000) Nature 403, 383-384; Chen MS et al (2000) Nature 403, 434-439; GrandPre T et al (2000) Nature 403,439-444; US005250414A; WO200005364A1; WO0031235).

Myelin-associated glycoprotein is a cell surface transmembrane moleculeexpressed on the surface of myelin consisting of five extracellularimmunoglobulin domains, a single transmembrane domain and anintracellular domain. MAG expression is restricted to myelinating gliain the CNS and peripheral nervous system (PNS). MAG is thought tointeract with neuronal receptor(s) which mediate effects on the neuronalcytoskeleton including neurofilament phosphorylation and inhibition ofneurite outgrowth in vitro. Although antagonists of MAG have beenpostulated as useful for the promotion of axonal sprouting followinginjury (WO9522344, WO9701352 and WO9707810), these claims are notsupported by in vivo data. Furthermore, the role of MAG as an inhibitorof axonal sprouting from CNS neurons in vivo is not proven (Li C M et al(1994) Nature 369, 747-750; Montag, D et al (1994) Neuron 13, 229-246;Lassmann H et al (1997) GLIA 19, 104-110; Li C et al (1998) J. Neuro.Res. 51, 210-217; Yin X et al (1998) J. Neurosci. 18, 1953-1962; BartschU et al (1995) Neuron 15 1375-1381; Li M et al (1996) 46,404-414).

Antibodies typically comprise two heavy chains linked together bydisulphide bonds and two light chains. Each light chain is linked to arespective heavy chain by disulphide bonds. Each heavy chain has at oneend a variable domain followed by a number of constant domains. Eachlight chain has a variable domain at one end and a constant domain atits other end. The light chain variable domain is aligned with thevariable domain of the heavy chain. The light chain constant domain isaligned with the first constant domain of the heavy chain. The constantdomains in the light and heavy chains are not involved directly inbinding the antibody to antigen.

The variable domains of each pair of light and heavy chains form theantigen binding site. The variable domains on the light and heavy chainshave the same general structure and each domain comprises a framework offour regions, whose sequences are relatively conserved, connected bythree complementarity determining regions (CDRs) often referred to ashypervariable regions. The four framework regions largely adopt abeta-sheet conformation and the CDRs form loops connecting, and in somecases forming part of, the beta-sheet structure. The CDRs are held inclose proximity by the framework regions and, with the CDRs from theother domain, contribute to the formation of the antigen binding site.CDRs and framework regions of antibodies may be determined by referenceto Kabat et al (“Sequences of proteins of immunological interest” USDept. of Health and Human Services, US Government Printing Office,1987).

It has now been found that an anti-MAG monoclonal antibody, described(Poltorak et al (1987) Journal of Cell Biology 105, 1893-1899, DeBellardet al (1996) Mol. Cell Neurosci. 7, 89-101; Tang et al (1997) Mol. Cell.Neurosci. 9, 333-346; Torigoe K and Lundborg G (1997) Exp. Neurology150, 254-262) and commercially available (MAB1567 (Chemicon)) whenadministered either directly into the brain or intravenously followingfocal cerebral ischaemia in the rat (a model of stroke), providesneuroprotection and enhances functional recovery. Therefore anti-MAGantibodies provide potential therapeutic agents for both acuteneuroprotection as well as the promotion of functional recoveryfollowing stroke. This antibody is a murine antibody. Although murineantibodies are often used as diagnostic agents their utility as atherapeutic has been proven in only a few cases. Their limitedapplication is in part due to the repeated administration of murinemonoclonals to humans usually elicits human immune responses againstthese molecules. To overcome these intrinsic undesireable properties ofmurine monoclonals “altered” antibodies designed to incorporate regionsof human antibodies have been developed and are well established in theart. For example, a humanised antibody contains complementaritydetermining regions (“CDR's”) of non human origin and the majority ofthe rest of the structure is derived from a human antibody.

The process of neurodegeneration underlies many neurologicaldiseases/disorders including acute diseases such as stroke, traumaticbrain injury and spinal cord injury as well as chronic diseasesincluding Alzheimer's disease, fronto-temporal dementias (tauopathies),peripheral neuropathy, Parkinson's disease, Huntington's disease andmultiple sclerosis. Anti-MAG mabs therefore may be useful in thetreatment of these diseases/disorders, by both ameliorating the celldeath associated with these diseases/disorders and promoting functionalrecovery.

All publications, both journal and patent, disclosed in this presentspecification are expressly and entirely incorporated herein byreference.

BRIEF SUMMARY OF THE INVENTION

The invention provides an altered antibody or functional fragmentthereof which binds to and neutralises MAG and comprises one or more ofthe following CDR's. The CDR's are identified as described by Kabat(Kabat et al. (1991) Sequences of proteins of immunological interest;Fifth Edition; US Department of Health and Human Services; NIHpublication No 91-3242. CDRs preferably are as defined by Kabat butfollowing the principles of protein structure and folding as defined byChothia and Lesk, (Chothia et al., (1989) Conformations ofimmunoglobulin hypervariable regions; Nature 342, p 877-883) it will beappreciated that additional residues may also be considered to be partof the antigen binding region and are thus encompassed by the presentinvention.

Light Chain CDRs

CDR According to Kabat L1 KSSHSVLYSSNQKNYLA (SEQUENCE ID NO: 1) L2WASTRES (SEQUENCE ID NO: 2) L3 HQYLSSLT (SEQUENCE ID NO: 3)Heavy Chain CDRs

CDR According to Kabat H1 NYGMN (SEQUENCE ID NO: 4) H2 WINTYTGEPTYADDFTG(SEQUENCE ID NO: 5) H3 NPINYYGINYEGYVMDY (SEQUENCE ID NO: 6)

The present invention also relates to an antibody which binds to thesame epitope as an antibody having the CDRs described above. Competitiveinhibition assays are used for mapping of the epitopes on an antigen.Thus there is also provided an anti-MAG antibody (altered or unaltered)which competitvely inhibits the binding of the altered antibody havingthe CDRs described supra to MAG, preferably human MAG.

In a further aspect, the present invention provides an altered antibodyor functional fragment thereof which comprises a heavy chain variabledomain which comprises one or more CDR's selected from CDRH1, CDRH2 andCDRH3 and/or a light chain variable domain which comprises one or moreCDRs selected from CDRL1, CDRL2 and CDRL3.

The invention further provides an altered anti-MAG antibody orfunctional fragment thereof which comprises:

-   -   a) a heavy chain variable domain (V_(H)) which comprises in        sequence CDRH1, CDRH2 and CDRH3, and/or    -   b) a light chain variable domain (V_(L)) which comprises in        sequence CDRL1, CDRL2 and CDRL3

A further aspect of the invention provides a pharmaceutical compositioncomprising an altered anti-MAG antibody of the present invention orfunctional fragment thereof together with a pharmaceutically acceptablediluent or carrier.

In a further aspect, the present invention provides a method oftreatment or prophylaxis of stroke and other neurological diseases in ahuman which comprises administering to said human in need thereof aneffective amount of an anti-MAG antibody of the invention or functionalfragments thereof.

In another aspect, the invention provides the use of an anti-MAGantibody of the invention or a functional fragment thereof in thepreparation of a medicament for treatment or prophylaxis of stroke andother neurological diseases.

In a further aspect, the present invention provides a method ofinhibiting neurodegeneration and/or promoting functional recovery in ahuman patient afflicted with, or at risk of developing, a stroke orother neurological disease which comprises administering to said humanin need thereof an effective amount of an anti-MAG antibody of theinvention or a functional fragment thereof.

In a yet further aspect, the invention provides the use of an anti-MAGantibody of the invention or a functional fragment thereof in thepreparation of a medicament for inhibiting neurodegeneration and/orpromoting functional recovery in a human patient afflicted with, or atrisk of developing, a stroke and other neurological disease.

Other aspects and advantages of the present invention are describedfurther in the detailed description and the preferred embodimentsthereof.

DESCRIPTION OF THE FIGURES

FIG. 1: Sequence of a mouse/human chimeric anti-MAG antibody heavy chain(Seq ID No. 27).

FIG. 2: Sequence of a mouse/human chimeric anti-MAG antibody light chain(Seq ID No. 28).

FIG. 3: Sequence of a mouse/human chimeric anti-MAG antibody heavy chain(Seq ID No. 29).

FIG. 4: Chimeric anti-MAG antibody binds to rat MAG

FIG. 5 Humanised anti-MAG antibody sequences

FIG. 6: Humanised anti-MAG antibodies bind to rat MAG

FIG. 7: Humanised anti-MAG antibodies bind to rat MAG

FIG. 8: Humanised anti-MAG antibodies bind to human MAG

FIG. 9: Competition ELISA with mouse and humanised anti-MAG antibodiesMAG

DETAILED DESCRIPTION OF THE INVENTION

Anti-MAG Antibody

The altered antibody of the invention is preferably a monoclonalantibody (mAb) and is preferably chimeric, humanised or reshaped, ofthese humanised is particularly preferred.

The altered antibody preferably has the structure of a natural antibodyor fragment thereof. The antibody may therefore comprise a completeantibody, a (Fab¹)₂ fragment, a Fab fragment, a light chain dimer or aheavy chain dimer. The antibody may be an IgG1, IgG2, IgG3, or IgG4; orIgM; IgA, IgE or IgD or a modified variant thereof. The constant domainof the antibody heavy chain may be selected accordingly. The light chainconstant domain may be a kappa or lambda constant domain. Furthermore,the antibody may comprise modifications of all classes eg IgG dimers, Fcmutants that no longer bind Fc receptors or mediate Clq binding(blocking antibodies). The antibody may also be a chimeric antibody ofthe type described in WO86/01533 which comprises an antigen bindingregion and a non-immunoglobulin region. The antigen binding region is anantibody light chain variable domain or heavy chain variable domain.Typically the antigen binding region comprises both light and heavychain variable domains. The non-immunoglobulin region is fused at its Cterminus to the antigen binding region. The non-immunoglobulin region istypically a non-immunoglobullin protein and may be an enzyme, a toxin orprotein having known binding specificity. The two regions of this typeof chimeric antibody may be connected via a cleavable linker sequence.Immunoadhesins having the CDRS as hereinbefore described are alsocontemplated in the present invention.

The constant region is selected according to the functionality required.Normally an IgG1 will demonstrate lytic ability through binding tocomplement and/or will mediate ADCC (antibody dependent cellcytotoxicity). An IgG4 will be preferred if an non-cytototoxic blockingantibody is required. However, IgG4 antibodies can demonstrateinstability in production and therefore is may be more preferable tomodify the generally more stable IgG1. Suggested modifications aredescribed in EPO307434 preferred modifications include at positions 235and 237. The invention therefore provides a lytic or a non-lytic form ofan antibody according to the invention

-   -   In a preferred aspect the altered antibody is class IgG, more        preferably IgG1.

In preferred forms therefore the antibody of the invention is a fulllength non-lytic IgG1 antibody having the CDRs described supra. In mostpreferred forms we provide a full length non-lytic IgG1 antibody havingthe CDRs of SEQ.I.D.NO:13 and 16 and a full length non-lytic IgG1antibody having the CDRs of SEQ.I.D.NO: 15 and 18.

In a further aspect, the invention provides polynucleotides encodingCDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3. Preferred polynucleotidesequences are

Light Chain CDRs

CDR L1 AAGAGCAGCCACAGCGTGCTGTACAGCAGCAA CCAGAAGAACTACCTGGCC (SEQUENCE IDNO: 7) L2 TGGGCCAGCACCCGCGAGAGC (SEQUENCE IDS NO: 8) L3CACCAGTACCTGAGCAGCCTGACC (SEQUENCE ID NO: 9)Heavy Chain CDRs

CDR H1 AACTACGGCATGAAC (SEQUENCE ID NO: 10) H2TGGATCAACACCTACACCGGCGAGCCCACCTAC GCCGACGACTTCACCGGC (SEQUENCE ID NO:11) H3 AACCCCATCAACTACTACGGCATCAACTACGAG GGCTACGTGATGGACTAC (SEQUENCE IDNO: 12)

In a further aspect of the invention, there is provided a polynucleotideencoding a light chain variable region of an altered anti-MAG antibodyincluding at least one CDR selected from CDRL1, CDRL2 and CDRL3, morepreferably including all 3 CDRs in sequence.

In a further aspect of the invention, there is provided a polynucleotideencoding a heavy chain variable region of an altered anti-MAG anti bodyincluding at least one CDR selected from CDRH1, CDRH2 and CDRH3, morepreferably including all 3 CDRs in sequence.

In a particularly preferred aspect, the anti-MAG antibody of theinvention is a humanised antibody.

The invention therefore further provides a humanised antibody orfunctional fragment thereof that binds to and neutralises MAG whichcomprises a heavy chain variable region comprising one of the followingamino acid sequences:—

(SEQ ID No 13) QVQLVQSGSELKKPGASVKVSCKASGYTFT NYGMN WVRQAPGQGLEWMG WINTYTGEPTYADDFTG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR NP INYYGINYEGYVMDYWGQGTLVTVSS. (Sequence ID No 14)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARNPINYYGINYEGYVMDYWGQGTLVTVSS (sequence ID No 15)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFTGRFVFSLDTSVSTAYLQISSLKAEDTATYFCARNPINYYGINYEGYVMDYWGQGTLVTVSS

In a further aspect of the invention there is provided a humanisedantibody or functional fragment thereof which binds to MAG whichcomprises the heavy chain variable region of Sequence ID No 13, 14 or 15together with a light chain variable region comprising amino acidSequences, Sequence ID No 16, 17, 18, or 19:

(SEQ ID No 16) DIVMTQSPDSLAVSLGERATINC KSSHSVLYSSNQKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC HQYLSS LT FGQGTKLEIKRTV(SEQ ID No 17) DIVMTQSPDSLAVSLGERATINC KSSHSVLYSSNQKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTIINLQAEDVAVYYC HQYLSS LT FGQGTKLEIKRTV(SEQ ID No 18) DIVMTQSPDSLAVSLGERATINC KSSHSVLYSSNQKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLHTEDVAVYYC HQYLSS LT FGQGTKLEIKRTV(SEQ ID No 19) DIVMTQSPDSLAVSLGERATINC KSSHSVLYSSNQKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTIINLHTEDVAVYYC HQYLSS LT FGQGTKLEIKRTV

In a further aspect of the present invention there is provided ahumanised antibody comprising:

-   -   a heavy chain variable fragment comprising SEQ ID No13, 14 or 15        and a constant part or fragment thereof of a human heavy chain        and    -   a light chain variable fragment comprising SEQ ID No 16, 17, 18        or 19 and a constant part or fragment thereof of a human light        chain.    -   Ina preferred aspect the humanised antibody is class 1gG more        preferably 1 gG1.

Preferred antibodies of the invention comprise:

-   -   Heavy chain variable region comprising Seq ID No 13 and light        chain variable region comprising Seq ID No 16;    -   Heavy chain variable region comprising Seq ID No 13 and light        chain variable region comprising Seq ID No 17;    -   Heavy chain variable region comprising Seq ID No 13 and light        chain variable region comprising Seq ID No 18;    -   Heavy chain variable region comprising Seq ID No 13 and light        chain variable region comprising Seq ID No 19.    -   Heavy chain variable region Scomprising eq ID No 14 and light        chain variable region comprising Seq ID No 16;    -   Heavy chain variable region comprising Seq ID No 14 and light        chain variable region comprising Seq ID No 17;    -   Heavy chain variable region comprising Seq ID No 14 and light        chain variable region comprising Seq ID No 18;    -   Heavy chain variable region comprising Seq ID No 14 and light        chain variable region comprising Seq ID No 19.    -   Heavy chain variable region Scomprising eq ID No 15 and light        chain variable region comprising Seq ID No 16;    -   Heavy chain variable region comprising Seq ID No 15 and light        chain variable region comprising Seq ID No 17;    -   Heavy chain variable region comprising Seq ID No 15 and light        chain variable region comprising Seq ID No 18;    -   Heavy chain variable region comprising Seq ID No 15 and light        chain variable region comprising Seq ID No 19.

In a further aspect, the invention provides polynucleotides encoding theheavy chain variable region comprising Sequence ID Nos 13, 14 and 15 andlight chain variable regions comprising Sequence ID No 16, 17, 18 and19.

Preferred polynucleotide Sequence encoding the amino acid Sequence SEQID NO 13 is

(SEQ ID No 20) CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGTTTCCTGCAAGGCTTCTGGATACACCTTACT AACTACGGCATG AACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGA TGGATCAACACCTACACCGGCGAGCCCACCTACGCCGACGACTTCACCGGC CGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTACTGTGCGAGAAACCCCTCAACTACTACGGCATCAACTACGAGGGCTACGTGATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAPreferred polynucleotide sequence encoding the amino acid Sequence ID No14 is:

(SEQ ID No 21) CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAACTACGGCATGAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACACCTACACCGGCGAGCCCACCTACGCCGACGACTTCACCGGCCGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTAAAGGCTGAGGACACTGCCGTGTATTTCTGTGCGAGAAACCCCATCAACTACTACGGCATCAACTACGAGGGCTACGTGATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAPreferred polynucleotide sequence encoding the amino acid Sequence ID No15 is:

(SEQ ID No 22) CAGGTGCAGCTGGTGCAATCTGGGTCTGAGTTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAACTACGGCATGAACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACACCTACACCGGCGAGCCCACCTACGCCGACGACTTCACCGGCCGGTTTGTCTTCTCCTTGGACACCTCTGTCAGCACGGCATATCTGCAGATCAGCAGCCTAAAGGCTGAGGACACTGCCACCTATTTCTGTGCGAGAAACCCCATCAACTACTACGGCATCAACTACGAGGGCTACGTGATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAPreferred polynucleotide sequence encoding the amino acid Sequence ID No16 is:

(SEQ ID No 23) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGC AAGAGCAGCCACAGCGTGCTGTACAGCAGCA ACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTAC TGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGT CACCAGTACCTGAGCAGCCTGACCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGPreferred polynucleotide sequence encoding SEQ ID No 17 is:

(SEQ ID No 24) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGC AAGAGCAGCCACAGCGTGCTGTACAGCAGCA ACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTACTGGGCATCTACCCGGGAATCC GGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCATCAACCTGCAGGCTGAAGATGTGGCAGTTTATTACTGT CACCAGTACCTGAGCAGC CTGACCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGPreferred polynucleotide encoding SEQ ID No 18 is:

(SEQ ID No 25) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGC AAGAGCAGCCACAGCGTGCTGTACAGCAGCA ACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTAC TGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCACACCGAAGATGTGGCAGTTTATTACTGT CACCAGTACCTGAGCAGC CTGACCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGPreferred polynucleotide encoding SEQ ID No 19 is:

(SEQ ID No 26) GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGC AAGAGCAGCCACAGCGTGCTGTACAGCAGCA ACCAGAAGAACTACCTGGCCTGGTACCAGCAGAAACCAGGACAGCCTCCT AAGCTGCTCATTTAC TGGGCATCTACCCGGGAATCCGGGGTCCCTGACCG ATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCATCAACCTGCACACCGAAGATGTGGCAGTTTATTACTGT CACCAGTACCTGAGCAGC CTGACCTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTG

“Neutralising” refers to inhibition, either total or partial, of MAGfunction including its binding to neurones and inhibition of neuriteoutgrowth.

“Altered antibody” refers to a protein encoded by an alteredimmunoglobulin coding region, which may be obtained by expression in aselected host cell. Such altered antibodies include engineeredantibodies (e.g., chimeric, reshaped, humanized or vectored antibodies)or antibody fragments lacking all or part of an Immunoglobulin constantregion, e.g., Fv, Fab, or F(ab)₂ and the like.

“Altered immunoglobulin coding region” refers to a nucleic acid sequenceencoding altered antibody. When the altered antibody is a CDR-grafted orhumanized antibody, the sequences that encode the complementaritydetermining regions (CDRs) from a non-human immunoglobulin are insertedinto a first immunoglobulin partner comprising human variable frameworksequences. Optionally, the first immunoglobulin partner is operativelylinked to a second immunoglobulin partner.

“First immunoglobulin partner” refers to a nucleic acid sequenceencoding a human framework or human immunoglobulin variable region inwhich the native (or naturally-occurring) CDR-encoding regions arereplaced by the CDR-encoding regions of a donor antibody. The humanvariable region can be an immunoglobulin heavy chain, a light chain (orboth chains), an analog or functional fragments thereof. Such CDRregions, located within the variable region of antibodies(immunoglobulins) can be determined by known methods in the art. Forexample Kabat et al. (Sequences of Proteins of Immunological Interest,4th Ed., U.S. Department of Health and Human Services, NationalInstitutes of Health (1987)) disclose rules for locating CDRS. Inaddition, computer programs are known which are useful for identifyingCDR regions/structures.

“Second immunoglobulin partner” refers to another nucleotide sequenceencoding a protein or peptide to which the first immunoglobulin partneris fused in frame or by means of an optional conventional linkersequence (i.e., operatively linked). Preferably it is an immunoglobulingene. The second immunoglobulin partner may include a nucleic acidsequence encoding the entire constant region for the same (i.e.,homologous—the first and second altered antibodies are derived from thesame source) or an additional (i.e., heterologous) antibody of interest.It may be an immunoglobulin heavy chain or light chain (or both chainsas part of a single polypeptide). The second immunoglobulin partner isnot limited to a particular immunoglobulin class or isotype. Inaddition, the second immunoglobulin partner may comprise part of animmunoglobulin constant region, such as found in a Fab, or F(ab)₂ (i.e.,a discrete part of an appropriate human constant region or frameworkregion). Such second immunoglobulin partner may also comprise a sequenceencoding an integral membrane protein exposed on the outer surface of ahost cell, e.g., as part of a phage display library, or a sequenceencoding a protein for analytical or diagnostic detection, e.g.,horseradish peroxidase, β-galactosidase, etc.

The terms Fv, Fc, Fd, Fab, or F(ab)₂ are used with their standardmeanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, ColdSpring Harbor Laboratory, (1988)).

As used herein, an “engineered antibody” describes a type of alteredantibody, i.e., a full-length synthetic antibody (e.g., a chimeric,reshaped or humanized antibody as opposed to an antibody fragment) inwhich a portion of the light and/or heavy chain variable domains of aselected acceptor antibody are replaced by analogous parts from one ormore donor antibodies which have specificity for the selected epitope.For example, such molecules may include antibodies characterized by ahumanized heavy chain associated with an unmodified light chain (orchimeric light chain), or vice versa. Engineered antibodies may also becharacterized by alteration of the nucleic acid sequences encoding theacceptor antibody light and/or heavy variable domain framework regionsin order to retain donor antibody binding specificity. These antibodiescan comprise replacement of one or more CDRs (preferably all) from theacceptor antibody with CDRs from a donor antibody described herein.

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., Queen et al.,Proc. Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al.,Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may beone selected from a conventional database, e.g., the KABAT® database,Los Alamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody. A humanantibody characterized by a homology to the framework regions of thedonor antibody (on an amino acid basis) may be suitable to provide aheavy chain constant region and/or a heavy chain variable frameworkregion for insertion of the donor CDRs. A suitable acceptor antibodycapable of donating light chain constant or variable framework regionsmay be selected in a similar manner. It should be noted that theacceptor antibody heavy and light chains are not required to originatefrom the same acceptor antibody. The prior art describes several ways ofproducing such humanised antibodies—see for example EP-A-0239400 andEP-A-054951

“Reshaped human antibody” refers to an altered antibody in whichminimally at least one CDR from a first human monoclonal donor antibodyis substituted for a CDR in a second human acceptor antibody.Preferrably all six CDRs are replaced. More preferrably an entireantigen combining region (e.g., Fv, Fab or F(ab′)₂) from a first humandonor monoclonal antibody is substituted for the corresponding region ina second human acceptor monoclonal antibody. Most preferrably the Fabregion from a first human donor is operatively linked to the appropriateconstant regions of a second human acceptor antibody to form a fulllength monoclonal antibody.

A “vectored antibody” refers to an antibody to which an agent has beenattached to improve transport through the blood brain barrier (BBB).(Review see Pardridge; Advanced Drug Delivery Review 36, 299-321, 1999).The attachment may be chemical or alternatively the moeity can beengineered into the antibody. One example is to make a chimera with anantibody directed towards a brain capilliary endothelial cell receptoreg an anti-insulin receptor antibody or anti-transferrin receptorantibody (Saito et al (1995) Proc. Natl. Acad. Sci. USA 92 10227-31;Pardridge et al (1995) Pharm. Res. 12 807-816; Broadwell et al (1996)Exp. Neurol. 142 47-65; Bickel et al (1993) Proc Natl. Acad. Sci. USA90, 2618-2622; Friden et al (1996) J. Pharm. Exp. Ther. 278 1491-1498,U.S. Pat. Nos. 5,182,107, 5,154,924, 5,833,988, 5,527,527). Once boundto the receptor, both components of the bispecific antibody pass acrossthe BBB by the process of transcytosis. Alternatively the agent may be aligand which binds such cell surface receptors eg insulin, transferrinor low density lipoprotein (Descamps et al (1996) Am. J. Physiol.270H1149-H1158; Duffy et al (1987) Brain Res. 420 32-38; Dehouck et al(1997) J. Cell Biol. 1997 877-889). Naturally occuring peptides such aspenetratin and SynB1 and Syn B3 which are known to improve transportacross the BBB can also be used (Rouselle et al (2000) Mol. Pharm. 57,679-686 and Rouselle et al (2001) Journal of Pharmacology andExperimental Therapeutics 296, 124-131).

The term “donor antibody” refers to an antibody (monoclonal, orrecombinant) which contributes the amino acid sequences of its variableregions, CDRs, or other functional fragments or analogs thereof to afirst immunoglobulin partner, so as to provide the alteredimmunoglobulin coding region and resulting expressed altered antibodywith the antigenic specificity and neutralizing activity characteristicof the donor antibody.

The term “acceptor antibody” refers to an antibody (monoclonal, orrecombinant) heterologous to the donor antibody, which contributes all(or any portion, but preferably all) of the amino acid sequencesencoding its heavy and/or light chain framework regions and/or its heavyand/or light chain constant regions to the first immunoglobulin partner.Preferably a human antibody is the acceptor antibody.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRS, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883. For convenience the CDR's as defined by Kabat in SEQ IDNos 13-26 are underlined.

CDRs provide the majority of contact residues for the binding of theantibody to the antigen or epitope. CDRs of interest in this inventionare derived from donor antibody variable heavy and light chainsequences, and include analogs of the naturally occurring CDRs, whichanalogs also share or retain the same antigen binding specificity and/orneutralizing ability as the donor antibody from which they were derived.

A “functional fragment” is a partial heavy or light chain variablesequence (e.g., minor deletions at the amino or carboxy terminus of theimmunoglobulin variable region) which retains the same antigen bindingspecificity and/or neutralizing ability as the antibody from which thefragment was derived.

An “analog” is an amino acid sequence modified by at least one aminoacid, wherein said modification can be chemical or a substitution or arearrangement of a few amino acids (i.e., no more than 10), whichmodification permits the amino acid sequence to retain the biologicalcharacteristics, e.g., antigen specificity and high affinity, of theunmodified sequence. For example, (silent) mutations can be constructed,via substitutions, when certain endonuclease restriction sites arecreated within or surrounding CDR-encoding regions. The presentinvention contemplates the use of analogs of the antibody of theinvention. It is well known that minor changes in amino acid or nucleicacid sequences may lead eg to an allelic form of the original proteinwhich retains substantially similar properties. Thus analogs of theantibody of the invention includes those in which the CDRs in thehypervariable region of the heavy and light chains are at least 80%homologous, preferably at least 90% homologous and more preferably atleast 95% homologous to the CDRs as defined above as CDRH1, CDRH2,CDRH3, CDRL1, CDRL2 and CDRL3 and retain MAG neutralising activity.Amino acid sequences are are at least 80% homologous if they have 80%identical amino acid residues in a like position when the sequences arealigned optimally, gaps or insertions being counted as non-identicalresidues. The invention also contemplates analogs of the antibodies ofthe invention wherein the framework regions are at least 80%, preferablyat least 90% and more preferably at least 95% homologous to theframework regions set forth in Seq ID 1-5. Amino acid sequences are atleast 80% homologous if they have 80% identical amino acid residues in alike position when the sequences are aligned optimally, gaps orinsertions being counted as non-identical residues.

Analogs may also arise as allelic variations. An “allelic variation ormodification” is an alteration in the nucleic acid sequence. Suchvariations or modifications may be due to degeneracy in the genetic codeor may be deliberately engineered to provide desired characteristics.These variations or modifications may or may not result in alterationsin any encoded amino acid sequence.

The term “effector agents” refers to non-protein carrier molecules towhich the altered antibodies, and/or natural or synthetic light or heavychains of the donor antibody or other fragments of the donor antibodymay be associated by conventional means. Such non-protein carriers caninclude conventional carriers used in the diagnostic field, e.g.,polystyrene or other plastic beads, polysaccharides, e.g., as used inthe BIAcore [Pharmacia] system, or other non-protein substances usefulin the medical field and safe for administration to humans and animals.Other effector agents may include a macrocycle, for chelating a heavymetal atom, or radioisotopes. Such effector agents may also be useful toincrease the half-life of the altered antibodies, e.g., polyethyleneglycol.

A neutralising antibody specific for MAG has been described (Poltorak etal (1987) Journal of Cell Biology 105,1893-1899, DeBellard et al (1996)Mol. Cell Neurosci. 7, 89-101; Tang et al (1997) Mol. Cell. Neurosci. 9,333-346; Torigoe K and Lundborg G (1997) Exp. Neurology 150, 254-262)and is commercially available (MAB1567 (Chemicon)).

Alternatively, one can construct antibodies, altered antibodies andfragments, by immunizing a non-human species (for example, bovine,ovine, monkey, chicken, rodent (e.g., murine and rat), etc.) to generatea desirable immunoglobulin upon presentation with native MAG from anyspecies against which antibodies cross reactive with human MAG can begenerated, eg human or chicken. Conventional hybridoma techniques areemployed to provide a hybridoma cell line secreting a non-human mAb toMAG. Such hybridomas are then screened for binding using MAG coated to384- or 96-well plates, with biotinylated MAG bound to a streptavidincoated plate. or in a homogenous europium-APC linked immunoassay usingbiotinylated MAG.

A native human antibody can be produced in a human antibody mouse suchas the “Xenomouse” (Abgenix) where the mouse immunoglobulin genes havebeen removed and genes encoding the human immunoglobulins have beeninserted into the mouse chromosome. The mice are immunised as normal anddevelop an antibody reponse that is derived from the human genes. Thusthe mouse produces human antibodies obviating the need to humanize theafter selection of positive hybridomas. (See Green L. L., J ImmunolMethods 1999 Dec. 10;231(1-2): 11-23)

The present invention also includes the use of Fab fragments or F(ab′)₂fragments derived from mAbs directed against MAG. These fragments areuseful as agents protective in vivo. A Fab fragment contains the entirelight chain and amino terminal portion of the heavy chain; and anF(ab′)₂ fragment is the fragment formed by two Fab fragments bound bydisulfide bonds. Fab fragments and F(ab′)₂ fragments can be obtained byconventional means, e.g., cleavage of mAb with the appropriateproteolytic enzymes, papain and/or pepsin, or by recombinant methods.The Fab and F(ab′)₂ fragments are useful themselves as therapeutic orprophylactic, and as donors of sequences including the variable regionsand CDR sequences useful in the formation of recombinant or humanizedantibodies as described herein.

The Fab and F(ab′)₂ fragments can also be constructed via acombinatorial phage library (see, e.g., Winter et al., Ann. Rev.Immunol., 12:433-455 (1994)) or via immunoglobulin chain shuffling (see,e.g., Marks et al., Bio/Technology, 10:779-783 (1992), which are bothhereby incorporated by reference in their entirety.

Thus human antibody fragments (Fv, scFv, Fab) specific for MAG can beisolated using human antibody fragment phage display libraries. Alibrary of bacteriophage particles, which display the human antibodyfragment proteins, are panned against the MAG protein. Those phagedisplaying antibody fragments that bind the MAG are retained from thelibrary and clonally amplified. The human antibody genes are thenexicised from the specific bacteriophage and inserted into human IgGexpression constructs containing the human IgG constant regions to formthe intact human IgG molecule with the variable regions from theisolated bacteriophage specific for MAG.

The donor antibodies may contribute sequences, such as variable heavyand/or light chain peptide sequences, framework sequences, CDRsequences, functional fragments, and analogs thereof, and the nucleicacid sequences encoding them, useful in designing and obtaining variousaltered antibodies which are characterized by the antigen bindingspecificity of the donor antibody.

Taking into account the degeneracy of the genetic code, various codingsequences may be constructed which encode the variable heavy and lightchain amino acid sequences, and CDR sequences as well as functionalfragments and analogs thereof which share the antigen specificity of thedonor antibody. Isolated nucleic acid sequences, or fragments thereof,encoding the variable chain peptide sequences or CDRs can be used toproduce altered antibodies, e.g., chimeric or humanized antibodies, orother engineered antibodies when operatively combined with a secondimmunoglobulin partner.

Altered immunoglobulin molecules can encode altered antibodies whichinclude engineered antibodies such as chimeric antibodies and humanizedantibodies. A desired altered immunoglobulin coding region containsCDR-encoding regions that encode peptides having the antigen specificityof an anti-MAG antibody, preferably a high affinity antibody, insertedinto a first immunoglobulin partner (a human framework or humanimmunoglobulin variable region).

Preferably, the first immunoglobulin partner is operatively linked to asecond immunoglobulin partner. The second immunoglobulin partner isdefined above, and may include a sequence encoding a second antibodyregion of interest, for example an Fc region. Second immunoglobulinpartners may also include sequences encoding another immunoglobulin towhich the light or heavy chain constant region is fused in frame or bymeans of a linker sequence. Engineered antibodies directed againstfunctional fragments or analogs of MAG may be designed to elicitenhanced binding.

The second immunoglobulin partner may also be associated with effectoragents as defined above, including non-protein carrier molecules, towhich the second immunoglobulin partner may be operatively linked byconventional means.

Fusion or linkage between the second immunoglobulin partners, e.g.,antibody sequences, and the effector agent may be by any suitable means,e.g., by conventional covalent or ionic bonds, protein fusions, orhetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde,and the like. Such techniques are known in the art and readily describedin conventional chemistry and biochemistry texts.

Additionally, conventional linker sequences which simply provide for adesired amount of space between the second immunoglobulin partner andthe effector agent may also be constructed into the alteredimmunoglobulin coding region. The design of such linkers is well knownto those of skill in the art. In further aspects of the invention weprovide diabodies (bivalent or bispecific), triabodies, tetrabodies andother multivalent scFV protein species having one or more CDRs asdescribed supra that bind to and neutralise MAG function.

In still a further embodiment, the antibody of the invention may haveattached to it an additional agent. For example, the procedure ofrecombinant DNA technology may be used to produce an engineered antibodyof the invention in which the Fc fragment or CH2-CH3 domain of acomplete antibody molecule has been replaced by an enzyme or otherdetectable molecule (i.e., a polypeptide effector or reporter molecule).

The second immunoglobulin partner may also be operatively linked to anon-immunoglobulin peptide, protein or fragment thereof heterologous tothe CDR-containing sequence having the antigen specificity of anti-MAGantibody. The resulting protein may exhibit both anti-MAG antigenspecificity and characteristics of the non-immunoglobulin uponexpression. That fusion partner characteristic may be, e.g., afunctional characteristic such as another binding or receptor domain, ora therapeutic characteristic if the fusion partner is itself atherapeutic protein, or additional antigenic characteristics.

Another desirable protein of this invention may comprise a completeantibody molecule, having full length heavy and light chains, or anydiscrete fragment thereof, such as the Fab or F(ab′)₂ fragments, a heavychain dimer, or any minimal recombinant fragments thereof such as anF_(v) or a single-chain antibody (SCA) or any other molecule with thesame specificity as the selected donor mAb. Such protein may be used inthe form of an altered antibody, or may be used in its unfused form.

Whenever the second immunoglobulin partner is derived from an antibodydifferent from the donor antibody, e.g., any isotype or class ofimmunoglobulin framework or constant regions, an engineered antibodyresults. Engineered antibodies can comprise immunoglobulin (Ig) constantregions and variable framework regions from one source, e.g., theacceptor antibody, and one or more (preferably all) CDRs from the donorantibody. In addition, alterations, e.g., deletions, substitutions, oradditions, of the acceptor mAb light and/or heavy variable domainframework region at the nucleic acid or amino acid levels, or the donorCDR regions may be made in order to retain donor antibody antigenbinding specificity.

Such engineered antibodies are designed to employ one (or both) of thevariable heavy and/or light chains of the anti-MAG mAb or one or more ofthe heavy or light chain CDRs. The engineered antibodies may beneutralising, as above defined.

Such engineered antibodies may include a humanized antibody containingthe framework regions of a selected human immunoglobulin or subtype, ora chimeric antibody containing the human heavy and light chain constantregions fused to the anti-MAG antibody functional fragments. A suitablehuman (or other animal) acceptor antibody may be one selected from aconventional database, e.g., the KABAT® database, Los Alamos database,and Swiss Protein database, by homology to the nucleotide and amino acidsequences of the donor antibody. A human antibody characterized by ahomology to the framework regions of the donor antibody (on an aminoacid basis) may be suitable to provide a heavy chain constant regionand/or a heavy chain variable framework region for insertion of thedonor CDRs. A suitable acceptor antibody capable of donating light chainconstant or variable framework regions may be selected in a similarmanner. It should be noted that the acceptor antibody heavy and lightchains are not required to originate from the same acceptor antibody.

Desirably the heterologous framework and constant regions are selectedfrom human immunoglobulin classes and isotypes, such as IgG (subtypes 1through 4), IgM, IgA, and IgE. However, the acceptor antibody need notcomprise only human immunoglobulin protein sequences. For instance agene may be constructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding anon-immunoglobulin amino acid sequence such as a polypeptide effector orreporter molecule.

Preferably, in a humanized antibody, the variable domains in both humanheavy and light chains have been engineered by one or more CDRreplacements. It is possible to use all six CDRs, or variouscombinations of less than the six CDRS. Preferably all six CDRs arereplaced. It is possible to replace the CDRs only in the human heavychain, using as light chain the unmodified light chain from the humanacceptor antibody. Alternatively, a compatible light chain may beselected from another human antibody by recourse to the conventionalantibody databases. The remainder of the engineered antibody may bederived from any suitable acceptor human immunoglobulin.

The engineered humanized antibody thus preferably has the structure of anatural human antibody or a fragment thereof, and possesses thecombination of properties required for effective therapeutic use.

It will be understood by those skilled in the art that an engineeredantibody may be further modified by changes in variable domain aminoacids without necessarily affecting the specificity and high affinity ofthe donor antibody (i.e., an analog). It is anticipated that heavy andlight chain amino acids may be substituted by other amino acids eitherin the variable domain frameworks or CDRs or both.

In addition, the constant region may be altered to enhance or decreaseselective properties of the molecules of the instant invention. Forexample, dimerization, binding to Fc receptors, or the ability to bindand activate complement (see, e.g., Angal et al., Mol. Immunol,30:105-108 (1993), Xu et al., J. Biol. Chem, 269:3469-3474 (1994),Winter et al., EP 307,434B).

An altered antibody which is a chimeric antibody differs from thehumanized antibodies described above by providing the entire non-humandonor antibody heavy chain and light chain variable regions, includingframework regions, in association with immunoglobulin constant regionsfrom other species, preferably human for both chains.

Preferably, the variable light and/or heavy chain sequences and the CDRsof suitable donor mAbs, and their encoding nucleic acid sequences, areutilized in the construction of altered antibodies, preferably humanizedantibodies, of this invention, by the following process. The same orsimilar techniques may also be employed to generate other embodiments ofthis invention.

A hybridoma producing a selected donor mAb is conventionally cloned, andthe DNA of its heavy and light chain variable regions obtained bytechniques known to one of skill in the art, e.g., the techniquesdescribed in Sambrook et al., (Molecular Cloning (A Laboratory Manual),2nd edition, Cold Spring Harbor Laboratory (1989)). The variable heavyand light regions containing at least the CDR-encoding regions and thoseportions of the acceptor mAb light and/or heavy variable domainframework regions required in order to retain donor mAb bindingspecificity, as well as the remaining immunoglobulin-derived parts ofthe antibody chain derived from a human immunoglobulin are obtainedusing polynucleotide primers and reverse transcriptase. The CDR-encodingregions are identified using a known database and by comparison to otherantibodies.

A mouse/human chimeric antibody may then be prepared and assayed forbinding ability. Such a chimeric antibody contains the entire non-humandonor antibody V_(H) and V_(L) regions, in association with human Igconstant regions for both chains.

Homologous framework regions of a heavy chain variable region from ahuman antibody may be identified using computerized databases, e.g.,KABAT®, and a human antibody having homology to the donor antibody willbe selected as the acceptor antibody. A suitable light chain variableframework region can be designed in a similar manner.

A humanized antibody may be derived from the chimeric antibody, orpreferably, made synthetically by inserting the donor mAb CDR-encodingregions from the heavy and light chains appropriately within theselected heavy and light chain framework. Alternatively, a humanizedantibody can be made using standard mutagenesis techniques. Thus, theresulting humanized antibody contains human framework regions and donormAb CDR-encoding regions. There may be subsequent manipulation offramework residues. The resulting humanized antibody can be expressed inrecombinant host cells, e.g., COS, CHO or myeloma cells.

A conventional expression vector or recombinant plasmid is produced byplacing these coding sequences for the antibody in operative associationwith conventional regulatory control sequences capable of controllingthe replication and expression in, and/or secretion from, a host cell.Regulatory sequences include promoter sequences, e.g., CMV promoter, andsignal sequences, which can be derived from other known antibodies.Similarly, a second expression vector can be produced having a DNAsequence which encodes a complementary antibody light or heavy chain.Preferably this second expression vector is identical to the firstexcept insofar as the coding sequences and selectable markers areconcerned, so to ensure as far as possible that each polypeptide chainis functionally expressed. Alternatively, the heavy and light chaincoding sequences for the altered antibody may reside on a single vector.

A selected host cell is co-transfected by conventional techniques withboth the first and second vectors (or simply transfected by a singlevector) to create the transfected host cell of the invention comprisingboth the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The humanized antibody whichincludes the association of both the recombinant heavy chain and/orlight chain is screened from culture by appropriate assay, such as ELISAor RIA. Similar conventional techniques may be employed to constructother altered antibodies and molecules.

Suitable vectors for the cloning and subcloning steps employed in themethods and construction of the compositions of this invention may beselected by one of skill in the art. For example, the conventional pUCseries of cloning vectors, may be used. One vector, pUC19, iscommercially available from supply houses, such as Amersham(Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).Additionally, any vector which is capable of replicating readily, has anabundance of cloning sites and selectable genes (e.g., antibioticresistance), and is easily manipulated may be used for cloning. Thus,the selection of the cloning vector is not a limiting factor in thisinvention.

Similarly, the vectors employed for expression of the antibodies may beselected by one of skill in the art from any conventional vector. Thevectors also contain selected regulatory sequences (such as CMVpromoters) which direct the replication and expression of heterologousDNA sequences in selected host cells. These vectors contain the abovedescribed DNA sequences which code for the antibody or alteredimmunoglobulin coding region. In addition, the vectors may incorporatethe selected immunoglobulin sequences modified by the insertion ofdesirable restriction sites for ready manipulation.

The expression vectors may also be characterized by genes suitable foramplifying expression of the heterologous DNA sequences, e.g., themammalian dihydrofolate reductase gene (DHFR). Other preferable vectorsequences include a poly A signal sequence, such as from bovine growthhormone (BGH) and the betaglobin promoter sequence (betaglopro). Theexpression vectors useful herein may be synthesized by techniques wellknown to those skilled in this art.

The components of such vectors, e.g. replicons, selection genes,enhancers, promoters, signal sequences and the like, may be obtainedfrom commercial or natural sources or synthesized by known proceduresfor use in directing the expression and/or secretion of the product ofthe recombinant DNA in a selected host. Other appropriate expressionvectors of which numerous types are known in the art for mammalian,bacterial, insect, yeast, and fungal expression may also be selected forthis purpose.

The present invention also encompasses a cell line transfected with arecombinant plasmid containing the coding sequences of the antibodies oraltered immunoglobulin molecules thereof. Host cells useful for thecloning and other manipulations of these cloning vectors are alsoconventional. However, most desirably, cells from various strains of E.coli are used for replication of the cloning vectors and other steps inthe construction of altered antibodies of this invention.

Suitable host cells or cell lines for the expression of the antibody ofthe invention are preferably mammalian cells such as NS0, Sp2/0, CHO,COS, a fibroblast cell (e.g., 3T3), and myeloma cells, and morepreferably a CHO or a myeloma cell. Human cells may be used, thusenabling the molecule to be modified with human glycosylation patterns.Alternatively, other eukaryotic cell lines may be employed. Theselection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. See, e.g., Sambrook et al., citedabove.

Bacterial cells may prove useful as host cells suitable for theexpression of the recombinant Fabs of the present invention (see, e.g.,Plückthun, A., Immunol. Rev., 130:151-188 (1992)). However, due to thetendency of proteins expressed in bacterial cells to be in an unfoldedor improperly folded form or in a non-glycosylated form, any recombinantFab produced in a bacterial cell would have to be screened for retentionof antigen binding ability. If the molecule expressed by the bacterialcell was produced in a properly folded form, that bacterial cell wouldbe a desirable host. For example, various strains of E. coli used forexpression are well-known as host cells in the field of biotechnology.Various strains of B. subtilis, Streptomyces, other bacilli and the likemay also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the artare also available as host cells, as well as insect cells, e.g.Drosophila and Lepidoptera and viral expression systems. See, e.g.Miller et al, Genetic Engineering, 8:277-298, Plenum Press (1986) andreferences cited therein.

The general methods by which the vectors may be constructed, thetransfection methods required to produce the host cells of theinvention, and culture methods necessary to produce the altered antibodyof the invention from such host cell are all conventional techniques.Likewise, once produced, the antibodies of the invention may be purifiedfrom the cell culture contents according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like. Such techniques arewithin the skill of the art and do not limit this invention. Forexample, preparation of altered antibodies are described in WO 99/58679and WO 96/16990.

Yet another method of expression of the antibodies may utilizeexpression in a transgenic animal, such as described in U.S. Pat. No.4,873,316. This relates to an expression system using the animal'scasein promoter which when transgenically incorporated into a mammalpermits the female to produce the desired recombinant protein in itsmilk.

Once expressed by the desired method, the antibody is then examined forin vitro activity by use of an appropriate assay. Presently conventionalELISA assay formats are employed to assess qualitative and quantitativebinding of the antibody to MAG. Additionally, other in vitro assays mayalso be used to verify neutralizing efficacy prior to subsequent humanclinical studies performed to evaluate the persistence of the antibodyin the body despite the usual clearance mechanisms.

The therapeutic agents of this invention may be administered as aprophylactic or post injury, or as otherwise needed. The dose andduration of treatment relates to the relative duration of the moleculesof the present invention in the human circulation, and can be adjustedby one of skill in the art depending upon the condition being treatedand the general health of the patient.

The mode of administration of the therapeutic agent of the invention maybe any suitable route which delivers the agent to the host. Theantagonists and antibodies, and pharmaceutical compositions of theinvention are particularly useful for parenteral administration, i.e.,subcutaneously, intramuscularly, intravenously, or intranasally.

Therapeutic agents of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the antagonist orantibody of the invention as an active ingredient in a pharmaceuticallyacceptable carrier. In the prophylactic agent of the invention, anaqueous suspension or solution containing the engineered antibody,preferably buffered at physiological pH, in a form ready for injectionis preferred. The compositions for parenteral administration willcommonly comprise a solution of the antagonist or antibody of theinvention or a cocktail thereof dissolved in an pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., 0.9% saline, 0.3% glycine, and the like.These solutions are sterile and generally free of particulate matter.These solutions may be sterilized by conventional, well knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, etc. The concentration of the antagonist or antibody of theinvention in such pharmaceutical formulation can vary widely, i.e., fromless than about 0.5%, usually at or at least about 1% to as much as 15or 20% by weight and will be selected primarily based on fluid volumes,viscosities, etc., according to the particular mode of administrationselected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of an antagonist or antibodyof the invention. Similarly, a pharmaceutical composition of theinvention for intravenous infusion could be made up to contain about 250ml of sterile Ringer's solution, and about 1 to about 30 and preferably5 mg to about 25 mg of an engineered antibody of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown or will be apparent to those skilled in the art and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa.

It is preferred that the therapeutic agent of the invention, when in apharmaceutical preparation, be present in unit dose forms. Theappropriate therapeutically effective dose can be determined readily bythose of skill in the art. To effectively treat stroke and otherneurological diseases in a human, one dose of up to 700 mg per 70 kgbody weight of an antagonist or antibody of this invention should beadministered parenterally, preferably i.v. or i.m. (intramuscularly).Such dose may, if necessary, be repeated at appropriate time intervalsselected as appropriate by a physician.

The antibodies described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilization and reconstitution techniques can be employed.

In another aspect, the invention provides a pharmaceutical compositioncomprising anti-MAG antibody of the present invention or a functionalfragment thereof and a pharmaceutically acceptable carrier for treatmentor prophylaxis of stroke and other neurological diseases.

In a yet further aspect, the invention provides a pharmaceuticalcomposition comprising the anti-MAG antibody of the present invention ora functional fragment thereof and a pharmaceutically acceptable carrierfor inhibiting neurodegeneration and/or promoting functional recovery ina human patient suffering, or at risk of developing, a stroke or otherneurological disease.

The following examples illustrate the invention.

EXAMPLE 1 Anti-MAG Antibody in Stroke Model

Materials and Methods

Anti-MAG Monoclonal Antibody

Anti-MAG monoclonal antibody was mouse anti-chick MAG antibody MAB 1567obtained from Chemicon. The antibody has the following characteristics:

Antigen: myelin-associated glycoprotein (human, mouse, rat, bovine,chick, frog)

Isotype: IgG1

Neutralising ability: see DeBellard et al (1996) Mol. Cell. Neurosci. 7,89-101; Tang et al (1997) Mol. Cell. Neurosci. 9, 333-346; Torigoe K andLundborg G (1997) Exp. Neurology 150, 254-262.

Control IgG1 mab was purchased from R+D Systems.

Intra-Cerebral Ventricular Cannulation (for Study 1 Only)

Under halothane anaesthesia intra-cerebral ventricular (i.c.v.) cannulaewere positioned in the left lateral cerebral ventricle (coordinates: 1.6mm from the midline, 0.8 mm caudal from bregma, 4.1 mm from skullsurface, incisor bar—3.2 mm below zero according to Paxinos and Watson,1986) All rats were singly housed to avoid damage to the guide or dummycannula. 7 days following surgery, correct cannula placement wasverified by an intense drinking response to Angiotensin II (100 ng,Simpson, et al. 1978). Nine days later, animals underwent cerebralischaemia.

Transient Focal Cerebral Ischaemia

Transient (90 min) focal cerebral ischaemia was induced in male SpragueDawley rats, each weighing between 300-350 g. The animals were initiallyanaesthetised with a mixture of 5% halothane, 60% nitrous oxide and 30%oxygen, placed on a facemask and anaesthesia subsequently maintained at1.5% halothane. Middle cerebral artery occlusion (MCAO) was carried outusing the intraluminal thread technique as described previously (ZeaLonga, et. al., 1989). Animals were maintained normothermic throughoutthe surgical procedure, allowed to recover for 1 h in an incubator,before being singly housed. Only those animals with a neurological scoreof 31 h post-occlusion were included in the study (as assessed using a5-point scoring system: 0, no deficit; 1, contralateral reflex; 2,weakened grip; 3, circling; 4, immobile; 5, dead). Animals weremaintained for up to 1 week at which time animals were killed bytranscardial perfusion of 0.9% saline followed by 4% paraformaldehyde in100 mM phosphate buffer. The brains were post-fixed in 4%paraformaldehyde at 4° C. for 48 h at which time they were removed fromthe skulls and cut into 2 mm blocks using a rat brain matrix. The 2 mmsections were then paraffin embedded using a Shandon Citadel 1000 tissueprocessor, cut into 6 μm sections using a microtome and mounted onpoly-L-lysine coated slides. Sections were then processed for CresylFast Violet (CFV) staining.

Dosing Regime

Anti-MAG monoclonal antibody and mouse IgG1 isotype control antibodywere dialysed against sterile 0.9% sodium chloride overnight andconcentrated appropriately.

-   Study 1: Animals received 2.5 μg of anti-MAGmab or 2.5 μg mouse IgG1    i.c.v. 1, 24 and 72 h following MCAO (5 ul per dose).-   Study 2: Animals received 200 μg of anti-MAG mab or 200 μg mouse IgG    i.v. 1 and 24 following MCAO.    Investigator was blinded to the identity of each dosing solution.    Neurological Assessment

Prior to induction of cerebral ischaemia, rats for Study 1 receivedtraining in beam walking and sticky label test. Animals not reachingcriteria in both tests were excluded from further study. Followingtraining, the remainder of the animals were stratified according toperformance into two balanced groups. Throughout the neurologicalassessment, the investigators were blinded to the treatment group of theanimal.

Bilateral Sticky Label Test

The bilateral sticky label test (Schallert et al., PharmacologyBiochemistry and Behaviour 16: 455-462, (1983)) was used to assesscontralateral neglect/ipsilateral bias. This models tactile extinctionobserved in human stroke patients (Rose, et al. 1994). This test hasbeen described in detail previously (Hunter, et al., Neuropharmacology39: 806-816 (2000); Virley et al Journal of Cerebral Blood Flow &Metabolism, 20: 563-582 (2000)). Briefly, a round, sticky paper labelwas placed firmly around the hairless area of the forepaws with equalpressure with order of placement randomised (left, right). Trainingsessions were conducted for 6 days prior to MCAO, day 6 data wasutilised as the preoperative baseline (Day 0). Animals were given twotrials 24 and 7 d following MCAO, the data represents a mean of the twotrials. The latency to contact and remove the labels were recorded andanalysed using the logrank test (Cox, J. Royal Statistical Society B 34:187-220 (1972)).

Beam Walking

Beam walking was used as a measure of hind-limb and fore-limbco-ordination by means of distance travelled across an elevated 100 cmbeam (2.3 cm diameter, 48 cm off the floor) as previously described indetail (Virley et al Journal of Cerebral Blood Flow & Metabolism, 20:563-582 (2000)). Rats were trained to cross the beam from start tofinish. For testing, each rat was given 2 trials 24 h and 7 d followingMCAO, the data represents a mean of the two trials. Statistical analysiswas ANOVA followed by Student's t-test.

The 27-Point Neurological Score (Study-1)

This study consists of a battery of tests to assess neurological statusincluding, paw placement, visual forepaw reaching, horizontal bar,contralateral rotation, inclined plane, righting reflex, contralateralreflex, motility & general condition, as described previously (Hunter,et al. Neuropharmacology 39: 806-816 (2000)) with the addition of gripstrength measurements (scores 2 for good right fore-limb grip, 1 forweak grip). Total score=27 for normal animal.

For study 2 this test was modified further: Grip strength—normal scores3, good—2, weak—1, very weak—0; Motility—normal scores 4, excellent—3,very good—2, good—1, fair—0; General Condition—normal scores 4,excellent—3, very good—2, good—1, fair—0; Circling—none scores 5,favours one-side scores 4, large circle—3, medium circle—2, smallcircle—1, spinning—0). Total score=32 for a normal animal.

In both studies animals were tested 1, 24, 48 h and 7 d following MCAO,a healthy normal animal scores 27 or 32 respectively. Data are presentedas median values, Statistical analysis was Kruskil Wallis ANOVA.

Lesion Assessment

Study 1—For each animal, lesion areas were assessed in sections fromthree pre-determined levels in the brain (0, −2.0 and −6.0 mm fromBregma respectively). Neuronal damage was assessed using cresyl fastviolet staining and the area of damage measured using an Optimas 6.1imaging package. Data is expressed as mean area (mm²)±sem.

Study 2—For each animal, lesion areas were assessed in sections fromseven pre-determined levels in the brain (+3 mm to −8 mm w.r.t. Bregma).Neuronal damage was assessed using cresyl fast violet staining and thearea of damage measured using an Optimas 6.1 imaging package. Data isexpressed as mean area (mm²)±sem.

Results

Study 1—Intra-Cerebral Ventrical (i.c.v.) Administration of Anti-MAG Mab

Neurological Score

One hour following MCAO animals in both treatment groups showed markedimpairment in neurological score (median score 12 in each group). Therewas no significant difference between groups at this time. However, 24(p=0.02), 48 (p=0.005) h and 7 d (p=0.006) following MCAO animalstreated with anti-MAG mab (2.5 μg, 1, 24 and 72 h post-MCAO) showedsignificantly improved Total Neurological score compared to thosetreated with control IgG. Median neurological scores 24, 48 h and 7 dfollowing MCAO in the IgG₁ treated group were 15, 14 and 18 respectivelycompared to 19.5, 21.5 and 22 in the anti-MAG mab treated animals. Onfurther analysis of the individual behaviours comprising the totalscore, this significant improvement was mainly attributed to improvedperformance in the following tests: paw placement (24 h, p=0.045; 48 h,p=0.016; 7 d, p=0.008), grip strength (24 h, p=0.049 48 h, p=0.0495; 7d, p=0.243), motility (24 h, p=0.199; 48 h, p=0.012; 7 d, p=0.067),horizontal bar (24 h, p=0.065; 48 h, p=0.005; 7 d, p=0.016), inclinedplane (24 h, p=0.006; 48 h, p=0.006; 7 d, p=0.169), visual forepawreaching (48 h, p=0.049, 7 d, p=0.049) and the degree of circling (24 h,p=0.417; 48 h, p=0.034; 7 d, p=0.183).

Beam Walking

Prior to surgery all animals were trained to cross the beam (100 cm).Twenty four hours following surgery there was a significant impairmenton the distance travelled on the beam in both anti-MAG (50±18 cm) andIgG₁ (22±14 cm) treated animals compared to pre-operative values.Although not significant, anti-MAG treated animals showed markedimprovement over IgG₁ treated animals in that they travelled twice thedistance of IgG₁ treated animals 24 h following tMCAO. Seven daysfollowing surgery however, while both groups showed marked improvementover time, the performance of animals treated with IgG remainedsignificantly impaired compared to baseline (55±15 cm; p=0.005). Incontrast however 7 d following MCAO, animals treated with anti-MAG mab(2.5 μg 1, 24 and 72 h, i.c.v post MCAO) performance was notsignificantly different from baseline (75±15 cm; p=0.07). This datashows that anti-MAG mab treatment accelerated recovery of this beamwalking task compared to mouse IgG₁ treated controls.

Sticky Label

Prior to surgery, animals in each of the treatment groups rapidlycontacted and removed the labels from each forepaw, there was nosignificant difference in the groups prior to treatment (Table 1).Twenty-four hours and 7 d following MCAO the latency to contact the leftpaw in each of the treatment groups remained relatively unaltered, whilethat of the right was markedly increased. However there was nosignificant differences between removal times in anti-MAG and IgG₁treated animals. In addition 24 h following MCAO, the latency to removalfrom both the left and right forepaw was significantly increased in bothtreatment groups compared to baseline, however in anti-MAG treatedanimals the latency to removal from the left paw was significantlyshorter than that of IgG₁ treated animals (p=0.03). There was also atrend for reduced latency to removal from the right paw in anti-MAGtreated animals compared to those treated with IgG₁ (p=0.08) (Table 1).At 7 d there was some degree of recovery in IgG₁ treated animals in thatthe latency to removal times for each forepaw were reduced compared tothose at 24 h (Table 1). This data suggests that treatment of rats withanti-MAG mab accelerate the recovery in this sticky label test followingtMCAO.

TABLE 1 Sticky label data Contact Time (s) Removal Time (s) (Mean ± sem)(Mean ± sem) Left Right Left Right Day Treatment Forepaw Forepaw ForepawForepaw 0 Anti-MAG 2.4 ± 0.2 3.6 ± 0.5 12 ± 2 12 ± 2 0 IgG₁ 3.3 ± 0.64.2 ± 0.7 10 ± 1  9 ± 1 1 Anti-MAG 5.9 ± 3.7 109.6 ± 27.5  *61 ± 26  96± 26 1 IgG₁ 3.6 ± 0.5 71.8 ± 31.7 130 ± 21 156 ± 19 7 Anti-MAG 3.8 ± 1  36.4 ± 10.2  54 ± 23  80 ± 30 7 IgG₁ 2.8 ± 0.3  64 ± 28  23 ± 8  87 ± 20*p = 0.03 Anti-MAG v's IgG₁ using the logrank testLesion Area Measurements

Administration of anti-MAG mab i.c.v, significantly reduced lesion areain two of the three brain levels examined compared to those animalstreated with equal amounts of mouse IgG₁ when examined 7 days followingtMCAO (Table 2).

TABLE 2 Mean Lesion Area ± sem (mm²) 7d following tMCAO 0 mm wrt −2 mmwrt −6 mm wrt Treatment Bregma Bregma Bregma Anti-MAG mab *9 ± 2 ^($)4 ±3 ^(#)3 ± 1  (n = 8) Mouse IgG₁ 14 ± 1 12 ± 1 5 ± 1 (n = 9) *p = 0.02,^($)p = 0.03, ^(#)p = 0.06, anti-MAG v's IgG₁, One-way, unpairedStudents T-TestStudy 2—Intra-Venous (i.v.) AdministrationNeurological Score

One and 24 hours following MCAO animals in both groups showed markedimpairment in neurological score. There was no significant differencebetween groups at this time, median scores 24 h following anti-MAG maband IgG₁ treatment were 20 and 18 respectively (p=0.5). Forty-eighthours following MCAO, animals treated with anti-MAG mab (200 ug, i.v. 1and 24 h post-MCAO) showed significant improvement in paw placement(p=0.048) and grip strength (p=0.033). Seven days following the onset ofcerebral ischaemia animals treated with anti-MAG mab continued toimprove (paw placement p=0.041; grip strength, p=0.048; motility,p=0.05) and showed significant improvement in total neurological score(median score 25) compared to those treated with mouse IgG₁ (Medianscore 23, p=0.047).

Lesion Area Measurements

The anti-MAG antibody when administered i.v. MCAO significantly reducedlesion area at 5 out of 7 pre-determined brain levels (+3 to −8 mmw.r.t. Bregma) compared to isotype controls, when examined 7 d followingMCAO.

Brain level Mean lesion area ± Mean lesion area ± wrt SEM (mm²) - SEM(mm²) - Bregma Anti- MAG treated Mouse IgG₁ treated  3 mm *0.38 ± 0.27 1.77 ± 0.45  1 mm *5.82 ± 1.65 9.627 ± 1.14 −1 mm  8.98 ± 2.58 12.07 ±1.57 −2 mm  7.28 ± 1.92 10.04 ± 1.87 −4 mm *5.57 ± 1.06 10.38 ± 1.39 −6mm *1.36 ± 0.51  4.43 ± 1.95 −8 mm *0.27 ± 0.27  1.93 ± 0.56 *p < 0.05 -Unpaired, one-way Students T-testConclusions

An anti-MAG monoclonal antibody administered either directly into theCSF or intravenously following transient middle-cerebral arteryocclusion in the rat, both reduced the area of cell death and improvedfunctional recovery compared to control treated animals. The degree ofneuroprotection seen in these studies suggests that this effect can notbe attributed to axonal sprouting as this would not result in neuronalsparing. The improvement in functional recovery seen 24 and 48 hfollowing MCAO probably reflects the degree of neuroprotection offeredby this antibody compared to control treated animals. However, over timethe animals appear to improve further, suggesting that blocking MAGactivity can also enhance functional recovery over time.

The studies presented here provide evidence that blocking the actions ofMAG provide both neuroprotection and enhanced functional recovery in arat model of stroke, and therefore anti-MAG antibodies provide potentialtherapeutic agents for acute neuroprotection and/or the promotion offunctional recovery following stroke. The low amounts of antibodyadministered via the i.v route and the resulting low serum levels of theantibody would in turn suggest extremely low antibody concentrations inthe brain due to the constraints of the blood brain barrier for antibodypenetration. Surprisingly, however, this still resulted in both,neuroprotection and enhanced functional recovery being observed.Anti-MAG antibodies also have potential use in the treatment of otherneurological disorders where the degeneration of cells and or nervefibres is apparent such as spinal cord injury, traumatic brain injury,peripheral neuropathy, Alzheimer's disease, fronto-temporal dementias(tauopathies), Parkinson's disease, Huntington's disease and MultipleSclerosis. In the examples that follow the CDRs of the chimeric andhumanised antibodies disclosed therein are the CDRs of the antibody ofexample 1.

EXAMPLE 2 Chimeric Antibody

Altered antibodies include chimeric antibodies which comprise variableregions deriving from one species linked to constant regions fromanother species. Examples of mouse-human chimeric anti-MAGimmunoglobulin chains of the invention are provided in FIGS. 1, 2, and3. Mouse-human chimeras using human IgG1, IgG2, IgG3, IgG4, IgA, IgE,IgM, IgD constant regions may be produced, as may chimeras associatingthe mouse variable regions with heavy or light chain constant regionsfrom non-human species.

FIG. 1 (Seq ID No. 27) provides the amino acid sequence of a chimericimmunoglobulin heavy chain in which the murine anti-MAG heavy chainvariable region is associated with a functional immunoglobulin secretionsignal sequence, and with an altered form of the human IgG1 constantregion, in which Kabat residues 248 and 250 have been mutated to alaninein order to disable the effector functions of binding to FcγRI andcomplement protein C1q (Duncan, A. R. and Winter, G. Localization of theC1q binding site on antibodies by surface scanning. Nature 332, 738-740,1988. Duncan, A. R., Woolf, J. M., Partridge, L. J., Burton, D. R. andWinter, G. Localisation of the binding site for human FcR1 on IgG.Nature 332, 563-564, 1988). Such mutations are optionally made in orderto customise the properties of an altered antibody to achieve aparticular therapeutic effect—for example binding to and blocking thefunction of an antigen without activating lytic effector mechanisms.

FIG. 2 (Seq ID No. 28) provides the amino acid sequence of a chimericimmunoglobulin light chain in which the murine anti-MAG light chainvariable region is associated with a functional immunoglobulin secretionsignal sequence, and with the human kappa constant region.

Similarly, the anti-MAG variable regions may be associated withimmunoglobulin constant regions which lack mutations disabling effectorfunctions. FIG. 3 (Seq ID No. 29) provides the amino acid sequence of achimeric immunoglobulin heavy chain in which the murine anti-MAG heavychain variable region is associated with a functional immunoglobulinsecretion signal sequence, and with a wild-type form of the human IgG1constant region.

From the information provided in FIGS. 1 to 3, cDNA inserts encodingthese chimeric chains may be prepared by standard molecular biologytechniques well known to those skilled in the art. Briefly, the geneticcode is used to identify nucleotide codons encoding the desired aminoacids, creating a virtual cDNA sequence encoding the chimeric protein.If the cDNA insert is desired to be expressed in a particular organism,then particularly favoured codons may be selected according to knowncodon usage biases. The desired nucleotide sequence is then synthesisedby means of PCR amplification of a template comprising overlappingsynthetic oligonucleotides which, as a contig, represent the desiredsequence. The resulting product may also be modified by PCR ormutagenesis to attach restriction sites to facilitate cloning into asuitable plasmid for expression or further manipulations.

EXAMPLE 3 Chimeric Antibody Binds to Rat MAG in ELISA

Chimeric anti-MAG antibody containing the light and heavy chain CDRs ofthe invention was produced by transient transfection of CHO cells. Forthis, Transfast transfection reagent (Promega; E2431) was used andtransfections carried out according to manufactures instructions. Inbrief, ˜10⁶ CHO cells were plated out per well of 6-well culture plates.The following day mammalian expression vector DNA encoding theappropriate heavy or light chain were mixed at 1:1 ratio (5 μg totalDNA) in medium (Optimem1 with Glutamax; Gibco #51985-026). Transfasttransfection reagent was added and the solution transferred to wellswith confluent cell layers. After 1 h at 37° C. in the cell incubator,the DNA/Transfast mixture was overlaid with 2 ml Optimem medium and leftfor 48-72 h in the incubator. Supernatants were harvested, cleared bycentrifugation and passed through 0.2 μm filters. Antibody concentrationin CHO cell culture supernatant was determined by ELISA and estimated tobe around 0.5 μg/ml. For MAG binding, commercially available ratMAG-Fcwas used. Due to the fusion with human Fc bound chimeric antibodiescould not be detected using anti-human IgG secondary antibodies.Instead, anti-human kappa light chain-specific antibody was used. FIG. 4shows that this chimeric antibody binds to MAG even at 1/64 dilution. Anunrelated humanised antibody and culture supernatant from mocktransfected cells did not generate any signal in this assay.

Procedure:

ELISA microtiter plates (Nunc Maxisorp) were coated with 1 μg/ml ratMAG-Fc fusion protein (R&D systems; 538-MG) in PBS at 4° C. overnight.Plates were washed twice with PBS and then blocked with PBS/BSA (1% w/v)for 1 h at room temperature (RT). Culture supernatants from transientlytransfected CHO cells were passed through 0.2 μm filters and serialdiluted in PBS/BSA starting at neat supernatant to 1/64 dilution. Sampledilutions were left at RT for 1 h. Plates were then washed three timeswith PBS/Tween 20 (0.1% v/v). Detection antibody was goat anti-humankappa light chain specific-peroxidase conjugate (Sigma A-7164) dilutedat 1/2000 in PBS/BSA. The detection antibody was incubated for 1 h at RTand the plates washed as above. Substrate solution (Sigma Fast OPDP-9187) was added and incubated until appropriate colour development wasdetected and then stopped using 3M H₂SO₄. Colour development was read at490 nm.

EXAMPLE 4 Humanised Antibodies

Altered antibodies include humanised antibodies which comprise humanisedvariable regions linked to human constant regions. Examples of humanisedanti-MAG immunoglobulin chains of the invention are provided in FIG. 5.Humanised antibodies using human IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM,IgD constant regions may be produced.

FIG. 5 (Seq ID No: 30) provides an example of the amino acid sequence ofa humanised immunoglobulin heavy chain in which the humanised anti-MAGheavy chain variable region is associated with a functionalimmunoglobulin secretion signal sequence, and with an altered form ofthe human IgG1 constant region, in which Kabat residues 248 and 250 havebeen mutated to alanine in order to disable the effector functions ofbinding to FcγRI and complement protein C1q (Duncan, A. R. and Winter,G. Localization of the C1q binding site on antibodies by surfacescanning. Nature 332, 738-740, 1988. Duncan, A. R., Woolf, J. M.,Partridge, L. J., Burton, D. R. and Winter, G. Localisation of thebinding site for human FcR1 on IgG. Nature 332, 563-564, 1988). Suchmutations are optionally made in order to customise the properties of analtered antibody to achieve a particular therapeutic effect—for examplebinding to and blocking the function of an antigen without activatinglytic effector mechanisms.

FIG. 5 (Seq ID No. 31) also provides an example of the amino acidsequence of a humanised immunoglobulin light chain in which thehumanised anti-MAG light chain variable region is associated with afunctional immunoglobulin secretion signal sequence, and with the humankappa constant region.

Similarly, the anti-MAG variable regions may be associated withimmunoglobulin constant regions which lack mutations disabling effectorfunctions. FIG. 5 (Seq ID No. 32) provides the amino acid sequence of ahumanised immunoglobulin heavy chain in which the humanised anti-MAGheavy chain variable region is associated with a functionalimmunoglobulin secretion signal sequence, and with a wild-type form ofthe human IgG1 constant region.

From the information provided in FIG. 5, cDNA inserts encoding thesehumanised chains may be prepared by standard molecular biologytechniques well known to those skilled in the art. Briefly, the geneticcode is used to identify nucleotide codons encoding the desired aminoacids, creating a virtual cDNA sequence encoding the protein. If thecDNA insert is desired to be expressed in a particular organism, thenparticularly favoured codons may be selected according to known codonusage biases. The desired nucleotide sequence is then synthesised bymeans of PCR amplification of a template comprising overlappingsynthetic oligonucleotides which, as a contig, represent the desiredsequence. The resulting product may also be modified by PCR ormutagenesis to attach restriction sites to facilitate cloning into asuitable plasmid for expression or further manipulations.

EXAMPLE 5 Humanised anti-MAG Antibodies Bind to Rat and Human MAG inElisa

1) Direct Binding ELISA to Rat MAG-Fc Fusion Protein of NormalisedAmounts of Culture Supernatant for 9 Humanised Heavy and Light ChainCombinations

Humanised anti-MAG antibodies containing the light and heavy chain CDRsof the invention were produced by transient transfection of CHO cells.For this, Transfast transfection reagent (Promega; E2431) was used andtransfections carried out according to manufactures instructions. Inbrief, ˜10⁶ CHO cells were plated out per well of 6-well culture plates.The following day mammalian expression vector DNA encoding theappropriate heavy or light chain were mixed at 1:1 ratio (5 μg totalDNA) in medium (Optimem1 with Glutamax; Gibco #51985-026). Transfasttransfection reagent was added and the solution transferred to wellswith confluent cell layers. After 1 h at 37° C. in the cell incubator,the DNA/Transfast mixture was overlaid with 2 ml Optimem medium and leftfor 48-72 h in the incubator. Supernatants were harvested, cleared bycentrifugation and passed through 0.2 μm filters. 9 heavy and lightvariable chain combinations were produced from the sequences shown inthe table below and the IgG1 heavy chain constant regions werefunctional according to Seq.ID.

Seq ID No (V-regions) Description Alternative name 13 Humanised Vh BVh114 Humanised Vh BVh2 15 Humanised Vh BVh3 16 Humanised Vl CVl1 17Humanised Vl CVl2 18 Humanised Vl CVl3 19 Humanised Vl CVl4

Antibody concentration was determined by ELISA and the amounts ofsupernatant used in the assay normalised to a starting concentration of250 or 500 ng/ml (depending on concentration of culture supernatant). Asantigen, commercially available ratMAG-Fc was used (R&D Systems;538-MG). Due to the fusion of this antigen with human Fc, bound chimericantibodies could not be detected using general anti-human IgG secondaryantibodies. Instead, anti-human kappa light chain-specific antibody wasused. FIG. 6 shows that all 9 humanised antibodies examined here boundto rat MAG with very similar binding curves down to ˜4 ng/ml. Thechimeric antibody used as a reference showed binding characteristicsthat fell within the group of humanised antibodies examined here.Although not absolute, this may suggest that the affinities of thehumanised antibodies examined here lie very closely within the affinityrange of the non-humanised chimeric antibody used as a reference here.

Procedure

96-well Nunc Maxisorp plates were coated overnight at 4° C. with ratMAG-Fc fusion protein (1 μg/ml; R&D Systems; Cat.No. 538-MG) in PBS.Plates were washed twice with PBS containing Tween20 (0.1% v/v; PBST)and blocked with PBS containing BSA (1% w/v) for 1 h at room temperature(RT). Variable amounts of culture supernatants were serial diluted inblocking buffer and added to the blocked wells starting at approximately500 or 250 ng/ml. Antibody concentrations of supernatants were based onindependent assays measuring the amount of humanised antibody present ineach culture supernatant. Chimeric mouse-human (non-humanised) antibodywas also included as reference. Antibody samples were incubated 1 h atRT and plates then washed 3× with PBST. Secondary antibody (Goatanti-human light chain specific-peroxidase conjugate; Sigma A-7164) wasadded diluted 1/5000 in blocking buffer and incubated for 1 h at RT.Wells were washed three times as above and binding detected by addingsubstrate (OPD tablets dissolved according to instructions; SigmaP-9187). Colour development was monitored and the reaction stopped using3M H₂SO₄. Colour development was read at 490 nm.

2) Direct Binding ELISA to Rat MAG-Fc Fusion Protein of Two PurifiedHumanised Anti-MAG Antibody Heavy-Light Chain Combinations

Two humanised antibodies consisting of heavy and light chain variableregion combinations BVh1/CVl1 and BVh3/CVl3 (table FIG. 5) and a mutatedIgG1 constant region as exemplified by SEQ.I.D.NO:30 (which is BVh1/CVl1mutated IgG1, those skilled in the art can readily derive the sequencefor the BVh3/CVl3 equivalent) were produced by a scaled-up version ofthe transient transfection described in example 3 and purified usingprotein A affinity chromatography. Purified antibody material wasdialysed against PBS and the concentration determined by OD280 reading;Antibody concentrations were adjusted to 5000 ng/ml and used as serialdilutions in a rat MAG-Fc binding ELISA. FIG. 7 shows that purifiedantibody material binds rat MAG-Fc and that both heavy and light chainvariable region combinations tested here are extremely similar.

Method:

96-well Nunc Maxisorp plates were coated overnight at 4° C. with ratMAG-Fc fusion protein (2.5 μg/ml; R&D Systems; Cat.No. 538-MG) in PBS.Plates were washed twice with PBS containing Tween20 (0.1% v/v; PBST)and blocked with PBS containing BSA (1% w/v) for 1 h at room temperature(RT). Purified humanised antibody was adjusted to a startingconcentration of 5 μg/ml in blocking buffer and then serial diluted.Antibody samples were incubated 1 h at RT and plates then washed 3× withPBST. Secondary antibody (Goat anti-human light chainspecific-peroxidase conjugate; Sigma A-7164) was added diluted 1/5000 inblocking buffer and incubated for 1 h at RT. Wells were washed threetimes as above and binding detected by adding substrate (OPD tabletsdissolved according to instructions; Sigma P-9187). Colour developmentwas monitored and the reaction stopped using 3M H₂SO₄ Colour developmentwas read at 490 nm.

Results:

Both purified humanised antibodies carrying none or several frameworkmutations show extremely similar binding to rat MAG. The results areseen in FIG. 7.

3) Binding to Human MAG Expressed on CHO Cells of Normalised Amounts ofCulture Supernatant for Two Humanised Heavy and Light Chain Combinations

The same humanised variable heavy and light chain combinations describedin example 52) were tested as cleared culture supernatants against humanMAG expressed on the surface of CHO cells. The amount of culturesupernatant used for each antibody was normalised based on antibodyconcentrations determined by ELISA. For this, 96-well plates (NuncMaxisorp) were coated overnight at 4° C. with goat anti-human IgG(gamma) chain (Sigma I-3382; in bicarbonate buffer pH9.6; 2 μg/ml).Following day, plates were washed twice with wash buffer (PBST) andblocked by adding at least 75 μl blocking buffer (PBS containing BSA 1%w/v) for 1 h at RT. Antibody sample solution were serial diluted inblocking buffer (starting dilution neat or ½) in duplicate. Ab standardwas purified humanised IgG1 antibody of an unrelated specificity andknown concentration.

The standard solution was also serial diluted across plate starting at500 ng/ml. All antibody solutions were incubated for 1 h at RT. Plateswere washed 3× as above and then incubated with goat anti-human light(kappa) chain specific (free and bound) peroxidase conjugate (Sigma;A-7164) at 1/5000 in blocking buffer for 1 h @ RT. Plates were againwashed 3× as above and incubated with substrate solution (OPD tablets;Sigma P-9187 until strong colour development. Colour development wasstopped by adding 25 μl 3M H2SO4 and the plate read at 490 nm.

FIG. 8 shows that both antibodies tested here are recognising human MAGand are very similar in their binding characteristics. CHO/− arenegative controls of CHO cells with no MAG expressed.

Method for Eu Cell-Based ELISA

96-well plates (Costar 3595) were filled with 100 μl cellsuspension/well (see table below for recommended cell number forperforming assay on days 1, 2, 3 or 4).

Day cell number/ml 1 3 × 105 2 1 × 105 3 5 × 104 4 1 × 104

Culture medium was removed and plates blocked with DMEM/F12 (SigmaD6421) containing FCS (10%), BSA (1%), NaN3 (1%; blocking buffer) for 1hour at RT. Blocking solution was then removed and sample added (inblocking buffer 50 μl/well). Incubated samples at 4° C. for 1 h. Plateswere then washed 3× with PBS using a Skatron plate washer. After wash,cells were fixed with 0.5% paraformaldehyde (diluted in PBS) for 20minutes at 4° C. and again washed as above. 50 μl/wellEuropium-conjugated secondary antibody diluted in Europium buffer (50 mMTris base, 150 mM NaCl, 0.5% BSA, 0.1 g/l, Tween 20, 7.86 mg/l DTPA atpH 7.3) was added and incubated for 1 h at 4° C.

Washed plates as above and added 200 μl Delphia enhancementsolution/well. Incubated solution at RT for 5-10 minutes. Wells wereread within 24 hours on a Victor.

4) Competition ELSA for Binding to Rat MAG-Fc Fusion Protein of TwoPurified Humanised Antibodies and the Non-Humanised Mouse MonoclonalAntibody

Method:

96-well Nunc Maxisorp plates were coated overnight at 4° C. with ratMAG-Fc fusion protein (2.5 μg/ml; R&D Systems; Cat.No. 538-MG) in PBS.Plates were washed twice with PBS containing Tween20 (0.1% v/v; PBST)and blocked with PBS containing BSA (1% w/v) for 1 h at room temperature(RT). Purified humanised antibody was adjusted to a concentration of 200ng/ml and mixed at equal volume with competitor molecules made up inblocking buffer ranging from 6000 to 0 ng/ml. Competitors were eitherparental mouse monoclonal antibody (anti-MAG) or an unrelated mousemonoclonal antibody (INN1) at the same concentrations (BVh1/CVl1 only).Antibody/competitor solutions were incubated 1 h at RT and plates thenwashed 3× with PBST. Secondary antibody (Goat anti-human light chainspecific-peroxidase conjugate; Sigma A-7164) was added diluted 1/5000 inblocking buffer and incubated for 1 h at RT. Wells were washed threetimes as above and binding detected by adding substrate (OPD tabletsdissolved according to instructions; Sigma P-9187). Colour developmentwas measured at 490 nm.

Results:

Both purified antibody preparations are equally competed by the originalmouse monoclonal antibody but not by a mouse monoclonal antibody thathas an unrelated specificity—see FIG. 9. This shows that the originalmouse monoclonal antibody and the humanised antibodies tested here areprobably recognising the same epitope and possibly have very similaraffinities to rat MAG.

1. A humanised anti-MAG antibody which binds to and neutralizes MAGcomprising, a heavy chain variable domain selected from the groupconsisting of: SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, and alight chain variable domain selected from the group consisting of: SEQID No. 16, SEQ ID No. 17, SEQ ID No. 18, and SEQ ID No.
 19. 2. Apharmaceutical composition comprising a humanised anti-MAG antibodywhich binds to and neutralizes MAG according to claim 1, furthercomprising a pharmaceutically acceptable diluent or carrier.
 3. Thehumanised anti-MAG antibody which binds to and neutralizes MAG accordingto claim 1, further comprising a constant part or fragment thereof of ahuman light chain and a constant part or fragment thereof of a humanheavy chain.
 4. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.13 and a light chain variable region of SEQ ID No.
 16. 5. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 13 and a light chain variable regionof SEQ ID No.
 17. 6. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.13 and a light chain variable region of SEQ ID No.
 18. 7. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 13 and a light chain variable regionof SEQ ID No.
 19. 8. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.14 and a light chain variable region of SEQ ID No.
 16. 9. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 14 and a light chain variable regionof SEQ ID No.
 17. 10. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.14 and a light chain variable region of SEQ ID No.
 18. 11. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 14 and light chain variable regionof SEQ ID No.
 19. 12. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.15 and a light chain variable region of SEQ ID No.
 16. 13. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 15 and a light chain variable regionof SEQ ID No.
 17. 14. A humanised anti-MAG antibody which binds to andneutralizes MAG comprising: a heavy chain variable region of SEQ ID No.15 and a light chain variable region of SEQ ID No.
 18. 15. A humanisedanti-MAG antibody which binds to and neutralizes MAG comprising: a heavychain variable region of SEQ ID No. 15 and a light chain variable regionof SEQ ID No. 19.